Effects of calcium concentration on nonviral gene delivery to bone marrow-derived stem cells

Abstract

Background: Calcium ions (Ca²⁺ ) influence natural bone healing, and calcium is frequently used in bone tissue engineering scaffolds and cements. Scaffolds can also incorporate gene delivery systems to further promote osteoblast differentiation. Thus, our goal was to identify if Ca²⁺ concentration affects the transfection of bone marrow stromal cells because these cells play a major role in bone healing and can infiltrate gene-activated scaffolds designed to promote bone growth.

Methods: Bone marrow-derived mesenchymal stem cells (BMSCs) were cultured in media with Ca²⁺ concentrations ranging from 0 to 20 mM and transfected with polyethyleneimine-plasmid DNA (PEI-pDNA) complexes. Cell viability and transfection efficiency were determined using MTS assays and flow cytometry, respectively. PEI-pDNA complex localization in BMSCs was assessed using fluorescence microscopy. To determine BMSC differentiation, messenger RNA (mRNA) for osteocalcin and CBFA1 was quantified using real time-polymerase chain reaction (PCR). Calcium deposition was qualitatively assessed after three and 14 days using Alizarin Red staining.

Result: Our results indicate that Ca²⁺ levels between 8 and 12 mM positively impacted transfection of BMSCs with PEI-pDNA complexes in terms of cell viability and transfection efficiency. A Ca²⁺ concentration of 10 mM also increased the expression of an osteogenic gene, osteocalcin, when the cells were transfected with plasmid DNA encoding bone morphogenetic protein 2 (BMP-2).

Conclusion: Ca²⁺ at a 10 mM concentration can significantly reduce toxicity and enhance transfection efficiency when combined with PEI-pDNA complexes, and this combination can be specifically applied to further enhance the differentiation of BMSCs by using the combination of polyethyleneimine-plasmid bone morphogenetic protein 2 (PEI-pBMP-2) and 10 mM Ca²⁺ as compared with PEI-pBMP-2 alone.

Keywords: BMP-2; bone tissue engineering; calcium ions; mesenchymal stem cell; non-viral gene delivery; polyethyleneimine-plasmid DNA.